Horseradish peroxidase is a frequently used enzyme in diagnostic tests based on enzyme-immunological reactions. Among these, one may mention pregnancy tests and tests for the detection of antiviral antibodies, such as hepatitis, human immuno-deficiency virus (HIV) and rubella.
Peroxidase, whether coupled or not to immunological carrier reagents, is unstable in diluted form in aqueous solution. This compels the test reagent manufacturer to supply the enzyme as a dry powder or in a concentrated form in a aqueous medium containing stabilizing agents such as bovine albumin or serum, whose concentrations may be as high as 20% of the total reagent volume. The user is thus required to carry out a dilution of the reagent at the moment of use, which may result in error and additional costs and is in any case inconvenience.
It is therefore advantageous to find stabilizing substances that allow the production of peroxidase and peroxidase-coupled reagents in stable diluted form in an aqueous medium, ready to use, with the optional additional requirement of being effective at a pH near neutrality.
Various investigations aimed at identifying stabilizing agents for peroxidase have been made, resulting in the following patents:
DE 3509238, corresponding to EPA 0 196 518 from Boehringer Mannheim PA1 DE 3511 327, corresponding to EPA 0 197 447 also from Boehringer, which discloses the incorporation of phenol or aminopyrine at a concentration equal or superior to 0.0005% PA1 CH 4944184, corresponding to EPA 0 070 992 from Hoffman-La Roche, claiming 4-amino-antipyrine; and PA1 USP 4,252,896 from Abbott disclosing the use of anilino-8-naphtalene-sulfonic acid.
None of these substances achieves satisfactory stabilization of peroxidase solutions.